This invention relates to compounds which are inhibitors of serine proteases and to pharmaceutical compositions thereof and their use in the treatment of the human or animal body.
The serine proteases are a group of proteolytic enzymes which have a common catalytic mechanism characterized by a particularly reactive Ser residue. Examples of serine proteases include trypsin, tryptase, chymotrypsin, elastase, thrombin, plasmin, kallikrein, Complement C1, acrosomal protease, lysosomal protease, cocoonase, xcex1-lytic protease, protease A, protease B, serine carboxypeptidase II, subtilisin, urokinase, Factor VIIa, Factor IXa, and Factor Xa. The serine proteases have been investigated extensively over a period of several decades and the therapeutic value of inhibitors of serine proteases is well understood.
Serine protease inhibitors play a central role in the regulation of a wide variety of physiological process including coagulation, fibrinolysis, fertilization, development, malignancy, neuromuscular patterning and inflammation. It is well known that these compounds inhibit a variety of circulating proteases as well as proteases that are activated or released in tissue. It is also becoming clear that serine protease inhibitors inhibit critical cellular processes, such as adhesion, migration, free radical production and apoptosis. In addition, animal experiments indicate that intravenously administered serine protease inhibitors, variants or cells expressing serine protease inhibitors, provide a protective effect against tissue damage.
Serine protease inhibitors have also been predicted to have potential beneficial uses in the treatment of disease in a wide variety of clinical areas such as oncology, neurology, haematology, pulmonary medicine, immunology, inflammation and infectious disease.
In particular serine protease inhibitors may be beneficial in the treatment of thrombotic diseases, asthma, emphysema, cirrhosis, arthritis, carcinoma, melanoma, restenois, atheroma, trauma, shock and reperfusion injury.
Thus for example an inhibitor of Factor Xa has value as a therapeutic agent as an anticoagulant, e.g. in the treatment and prevention of thrombotic disorders. The use of a Factor Xa inhibitor as an anticoagulant is desirable in view of the selectivity of its effect. Many clinically approved anticoagulants have been associated with adverse events owing to the non-specific nature of their effects on the coagulation cascade.
Also, there are well-known associations of al protease inhibitor deficiency with emphysema and cirrhosis and C1 esterase inhibitor deficiency with angioedema.
We have now found that certain novel amino substituted fused bicyclic compounds are particularly effective as inhibitors of serine proteases, especially proteases with negatively charged Pi specificity pockets, and most especially the serine proteases thrombin, trypsin, urokinase and Factor Xa. It is envisaged that compounds of the type described below will be readily bioavailable, particularly orally bioavailable.
Thus viewed from one aspect the invention provides serine protease inhibitor compounds of formula I 
(where R1 is hydrogen, halo, cyano, nitro or hydroxyl, amino, alkoxy, alkyl, aminoalkyl, hydroxyalkyl, thiol, alkylthio, aminosulphonyl, alkoxyalkyl, alkoxycarbonyl, acyloxymethoxycarbonyl or alkylamino optionally substituted by hydroxy, alkylamino, alkoxy, oxo, aryl, cycloalkyl, amino, halo, cyano, nitro, thiol, alkylthio, alkylsulphonyl, alkylsulphenyl, alkylsulphonamido, alkylaminosulphonyl, haloalkoxy and haloalkyl;
R2 is hydrogen, halo, methyl, amino, hydroxy or oxo; and
R is Xxe2x80x94Xxe2x80x94Y(R7)xe2x80x94Lxe2x80x94Lp(D)n
(wherein each X independently is a C, N, O or S atom or a CO, CR1, C(R)2 or NR1 group, at least one X being C, CO, CR or a C(R)2 group;
Y (the xcex1-atom) is a nitrogen atom or a CR1 group or Y and L taken together form a cyclic group;
R7 is a lipophilic group, e.g. alkyl, alkenyl, mono- or bi-cycloalkyl, aryl, heteroaryl, mono- or bicycloalkylalkyl, mono- or bicycloalkylalkenyl, aralkyl, heteroaryl-alkyl, arylalkenyl, heteroarylalkenyl all optionally substituted by a group R1;
L is an organic linker group containing 1 to 5 backbone atoms selected from C, N, O and S, or a branched alkyl or cyclic group;
Lp is a lipophilic organic group, e.g. an alkyl, heterocyclic, alkenyl, alkaryl, cycloalkyl, polycycloalkyl, cycloalkenyl, aryl, aralkyl or haloalkyl group or a combination of two or more such groups optionally substituted by one or more of oxa, thia, aza or R1 groups, preferably a group containing up to 25 carbon atoms;
D is a hydrogen bond donor group; and n is 0, 1 or 2);
or a physiologically tolerable salt thereof, e.g. a halide, phosphate or sulphate salt or a salt with ammonium or an organic amine such as ethylamine or meglumine.
In the compounds of the invention, unless otherwise indicated, aryl groups preferably contain 5 to 10 ring atoms optionally including 1, 2 or 3 heteroatoms selected from O, N and S; alkyl, alkenyl or alkynyl groups or alkylene moieties preferably contain up to 6 carbons; cyclic groups preferably have ring sizes of 3 to 8 atoms; and fused multicyclic groups preferably contain 8 to 16 ring atoms.
In the compounds of the invention the fused ring system is preferably a 1-aminoisoquinoline system.
Particularly as a substituent on the isoquinoline ring, R1 is preferably hydrogen, hydroxy, amino or alkyl. R2 is preferably hydrogen. Two or more non-hydrogen R1 (or R2) groups may be present on the carbocyclic (or heterocyclic) rings; however a single non-hydrogen R1 (or R2) group is preferred. R1 is preferably on the 6-position of the fused ring system.
R is depicted in formula (I) as being present on the 7-position of the fused ring system although it is also envisaged that the lipophilic group could be present on the 6-position. However, preferably the lipophilic group will be present on the 7-position of the ring.
In the compounds of the invention, where the alpha atom (Y) is carbon it preferably has the conformation that would result from construction from a D-xcex1-aminoacid NH2xe2x80x94CR1(R7)xe2x80x94COOH. Likewise the fourth substituent R1 at an alpha carbon is preferably a methyl or hydroxymethyl group or most preferably hydrogen.
The linker group Xxe2x80x94X from the fused bicyclic group to the alpha atom is preferably selected from xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94CONR1xe2x80x94, xe2x80x94NHxe2x80x94COxe2x80x94, xe2x80x94NHxe2x80x94CH2xe2x80x94,xe2x80x94CH2xe2x80x94NHxe2x80x94, xe2x80x94CH2Oxe2x80x94, xe2x80x94OCH2xe2x80x94, xe2x80x94COOxe2x80x94, xe2x80x94OCxe2x95x90Oxe2x80x94 and xe2x80x94CH2CHR1xe2x80x94 (e.g. xe2x80x94CH2CH2xe2x80x94). Preferably, the X moiety nearest to the alpha atom is an NH or O atom, most preferably a NH group. The X moiety alpha to the fused ring system is preferably a carbon based group such as CH2 or CO, preferably CO. 1-amino-7-carbonylisoquinoline compounds optionally saturated between the 3 and 4 positions are novel and themselves form a further aspect of the invention. 1-amino-7-substituted isoquinoline compounds optionally saturated between the 3 and 4 positions also form an aspect of the invention.
R1 preferably represents an unsubstituted or R1 substituted aryl or cyclohexyl group, preferably phenyl or naphthyl.
The linker group from the alpha atom to the lipophilic group is preferably CO, CH2NH, CONR1(CH2)m, (CH2)mN(R1)CO(CH2)m, (CH2)m+2, (CH2)mCO(CH2)m, (CH2)mOCxe2x95x90O, (CH2)mO or CHxe2x95x90CH(CH2)m (where each m is independently 0 or 1). The linker may be optionally branched, for example, to incorporate a polar functionality. In one embodiment Y and L taken together form a cyclic group and the alpha atom is therefore a carbon atom. The cyclic group can be unsubstituted or substituted and can have a ring size of from 3 to 8 atoms. Preferably, the cyclic group is a cyclic amide, most preferably wherein the amide nitrogen of the cyclic amide group is bound to the lipophilic group.
The lipophilic group preferably comprises a cycloalkyl, azacycloalkyl, diazacycloalkyl, phenyl, naphthyl, adamantyl, decalinyl, tetrahydrodecalinyl, bicycloalkyl, mono- or diazabicycloalkyl, mono- or bicyclo heteroaromatic or a linear or branched alkyl, alkylene, alkenyl or alkenylene group all optionally substituted by oxa, aza, thia or one or more groups R1, or a combination of at least two such groups linked by a Spiro linkage or a single or double bond or by Cxe2x95x90O, O, S, SO, SO2, CONR1, NR1xe2x80x94COxe2x80x94, NR1 linkage. For example, representative lipophilic groups include methylcyclohexyl, methylcyclohexylmethyl, methylphenylmethyl, phenylethyl, benzylpiperidinyl, benzoylpiperidinyl, bispiperidinyl or phenylpiperazinyl.
Most preferably, the lipophilic group is selected from 
wherein
R3 is R1, aryl or cycloalkyl;
m represents 0 or 1;
R4 represents hydrogen, (CH2)wCOOH, (CH2)wCON(R1)2, (CH2)wCONxcex1-AminoAcid;
w represents an integer from 0 to 4; and
X represents CH or N.
For example specific lipophilic groups include 
especially when R8 represents H, OMe, F, NO2, OH or Cl.
The hydrogen bond donor group which may be attached to the lipophilic group preferably has a nitrogen or oxygen atom as the donor atom and conveniently is a hydroxyl group, a primary, secondary or tertiary amine, or a primary or secondary imine group (as part of an amidine or guanidine) or a saturated or unsaturated heterocyclic group containing a ring nitrogen, preferably a group containing 5 to 7 ring atoms. Where the donor atom is a ring nitrogen, the remote portion of the heterocyclic ring may be part of the lipophilic group.
Accordingly, preferred compounds of the invention are of formula 
(wherein R1 is as hereinbefore defined, R1 preferably being hydrogen, hydroxy or amino);
R5 and R6 are hydrogen or taken together with the carbon atom to which they are attached represent a carbonyl group;
Ar is an unsubstituted or R1 substituted aryl or cyclohexyl group, preferably phenyl or naphthyl;
Xxe2x80x94X is xe2x80x94CONHxe2x80x94, xe2x80x94CH2CH2xe2x80x94, CH2Oxe2x80x94, xe2x80x94COOxe2x80x94, xe2x80x94CH2NHxe2x80x94, xe2x80x94OCH2xe2x80x94 or xe2x80x94NHCH2xe2x80x94;
L1 is a valence bond or an organic linker group containing 1 to 4 backbone atoms selected from C, N and O;
Lp1 is a cycloalkyl, azacycloalkyl, diazacycloalkyl, phenyl, naphthyl, adamantyl, decalinyl, tetrahydrodecalinyl, bicycloalkyl, mono- or diazabicycloalkyl, mono- or bicyclo heteroaromatic or a linear or branched alkyl, alkylene, alkenyl or alkenylene group all optionally substituted by oxa, aza, thia or a group R1, or a combination of at least two such groups linked by a Spiro linkage or a single or double bond or by Cxe2x95x90O, O, S, SO, SO2, CONR1, NR1xe2x80x94COxe2x80x94, NR1 linkage. For example, representative lipophilic groups include a methyl-cyclohexyl, methylcyclohexylmethyl, methylphenylmethyl, phenylethyl, benzylpiperidinyl, benzoylpiperidinyl, bispiperidinyl or phenylpiperazinyl and those as hereinbefore described;
D is a hydrogen bond donor group;
and n is 0, 1 or 2).
In one embodiment, L1 comprises the backbone of an alpha amino acid, the lipophilic group being the side chain of the amino acid. The carboxyl part of the alpha amino acid may be optionally coupled via an amide bond to an amino acid or to a primary or secondary cyclic or acyclic alkyl amine or diamine or via an ester bond to primary or secondary alcohols.
In a preferred embodiment, L1 represents a valence bond and the lipophilic group is bound directly to the carbonyl alpha to the alpha atom via a nitrogen atom which forms part of the lipophilic group. Suitable lipophilic groups in this case therefore include piperidinyl, pyrrolidinyl and piperazinyl. In a preferred embodiment the piperidine or piperazinyl group is further substituted by a phenyl, benzyl, benzoyl, pyridyl, pyridyloxy, piperidinyl or phenoxy group, optionally substituted by one or more R1 groups. Where the lipophilic group is pyridyl it is envisaged then N-oxide pyridyl compounds will also be effective.
In a further embodiment, the lipophilic group has attached a group of the formula xe2x80x94COOR1, CON(R1)2 or xe2x80x94CONxcex1-aminoacid or ester derivative thereof.
In another embodiment the group binding the alpha carbon atom to the lipophilic group comprises a heterocyclic group. Accordingly, preferred compounds of the invention also include 
(wherein R1 is as hereinbefore defined R1 preferably being hydrogen, hydroxy or amino);
Ar is an unsubstituted or R1 substituted aryl or cyclohexyl group, preferably phenyl or naphthyl;
Xxe2x80x94X is xe2x80x94CONHxe2x80x94, xe2x80x94CH2CH2xe2x80x94, CH2Oxe2x80x94, xe2x80x94COOxe2x80x94, xe2x80x94CH2NHxe2x80x94, xe2x80x94OCH2xe2x80x94 or xe2x80x94NHCH2xe2x80x94;
m is 0, 1 or 2;
Het is a 5 or 6-membered heterocyclic group interrupted by 1, 2 or 3 heteroatoms selected from O, N and S optionally substituted by a group R1;
Lp1 is a cycloalkyl, azacycloalkyl, diazacycloalkyl, phenyl, naphthyl, adamantyl, decalinyl, tetrahydrodecalinyl, bicycloalkyl, mono- or diazabicycloalkyl, mono- or bicyclo heteroaromatic or a linear or branched alkyl, alkylene, alkenyl or alkenylene group all optionally substituted by a group R1, or a combination of at least two such groups linked by a spiro linkage or a single or double bond or by Cxe2x95x90O, O, S, SO, SO2, CONR1, NR1xe2x80x94COxe2x80x94, NR1 linkage; for example representative lipophilic groups include a cyclohexyl, phenyl, benzyl, benzyloxy or benzoyl;
D is a hydrogen bond donor group;
and n is 0, 1 or 2).
Where Het is a five membered ring, the two ring atoms at which it is connected are preferably separated by one ring atom. Where Het is a six-membered ring, the two ring atoms at which it is connected are preferably separated by one or two ring atoms. Representative heterocyclic groups include thiazole, oxazole, oxadiazole, triazole, thiadiazole or imidazole. Where the heterocyclic group is substituted by R1 this is preferably a COOH, CON(RI)2 or COOR1 connected to the heterocycle via a valence bond or alkylene chain.
In a further embodiment, the lipophilic group has attached a group of the formula xe2x80x94COOR1 or xe2x80x94CONxcex1-aminoacid or ester derivative thereof.
Especially preferred compounds according to the invention include 1-Aminoisoquinolin-7-oyl-D-phenylglycinyl-D-2-naphthylalaninylproline, 1-Aminoisoquinolin-7-oyl-D-phenylglycine-4-(4xe2x80x2-pyridyl) piperidinamide, 1-Aminoisoquinolin-7-oyl-D-phenylglycine-4-methoxybenzylamide, 1-Aminoisoquinolin-7-oyl-D-phenylglycine-4-(4xe2x80x2-hydroxyphenyl)piperazinamide, 1-Aminoisoquinolin-7-oyl-D-phenylglycine-4-(2,4-difluorophenyl) piperazinamide, 1-Aminoisoquinolin-7-oyl-D-cyclohexylglycine-4-(4xe2x80x2-pyridyl)piperazinamide, 1-Aminoisoquinolin-7-oyl-D,L-1-naphthylglycine-4,4-bipiperidinamide, 2(R)-{[N-(1-Aminoisoquinoline)-7-oyl]amino}-2-phenylethyl-4-methylbenzamide and 1-Aminoisoquinolin-7-oyl-D-phenylglycine-4,4xe2x80x2-(1xe2x80x2-methyl)bipiperidinamide.
The use of aminoisoquinolines, particularly 1-amino-isoquinolines, more particulary 6 and/or 7 substituted isoquinolines, especially 7-substituted-1,6-diamino-isoquinolines, as serine protease inhibitors, especially factor Xa inhibitors, in particular in therapeutic or prophylactic treatments of humans or non-human animals, particularly mammals, is novel and forms a further aspect of the present invention.
The compounds of the invention may be prepared by conventional chemical synthetic routes, e.g. by amide bond formation to couple the fused bicyclic group to the. lipophilic group. A preferred fused bicyclic group for coupling to the lipophilic group is 1-amino-7-carboxyisoquinoline which can be prepared from the readily avaliable starting material 7-carboxyisoquinoline (F. T. Tyson, JACS, 1939,61,183). Amination of the 7-carboxyisoquinoline can be readily effected using ammonia as described in the Examples below.
Preparation of the lipophilic group is conveniently effected from for example LpNH2 (where Lp is as hereinbefore defined). LpNH2 may be attached to a resin to allow routine solid phase peptide synthesis to be carried out. The resin attached LpNH2 moiety can then be coupled by conventional techniques to a suitably protected amino acid, whose side chain will form the R7 group, via for example a free amino group in the Lp group. Deprotection can then be effected before coupling to 1-amino-7-carboxyisoquinoline and isolation from the resin. This method is illustrated in Scheme 1 below.
Alternatively, the synthesis can be initiated from an LpCOOH derivative and the carboxyl functionality can be coupled to the resin. Conventional peptide synthesis as illustrated in Scheme 2 can then be carried out.
Further peptide based syntheses are illustrated in Schemes 3 to 5. 
In all the syntheses illustrated in Schemes 1 to 5 the coupling reactions can of course be effected using an activated form of the carbonyl compound e.g. an acid chloride or active ester.
Where Y and L taken together form a cyclic amide group this can be conveniently introduced by reacting an Lp group carrying a secondary amine with an active side chain with a suitable amino acid. Cyclisation can be base induced via nucleophilic attack of the alpha atom on a leaving group on the active side chain.
If necessary the amide linkage from isoquinoline to alpha atom can be reduced using an appropriate reducing agent employing the necessary protection.
Alternatively, the compounds of the invention where Xxe2x80x94X is Cxe2x80x94N or Cxe2x80x94O can be prepared from compounds of formula H2NCH(R7)Rxe2x80x3 or HOCH(R7)Rxe2x80x3 respectively, by condensation with an appropriately substituted isoquinoline, e.g. 1-amino-7-bromomethyl-isoquinoline, in the presence of a base, where Rxe2x80x3 is Lxe2x80x94Lp(D)n or a functionality which can be converted to such by methods herein described.
Compounds of the invention where Xxe2x80x94X is Cxe2x80x94N can also be prepared from compounds of the formula H2NCH(R7)Rxe2x80x3 by condensation with an appropriately substituted isoquinoline aldehyde or ketone, e.g. 1-amino-isoquinoline-7-carboxaldehyde, in the presence of a reducing agent such as sodium borohydride or sodium triacetoxyborohydride.
Alternatively, the compounds of the invention be prepared from compounds of formula NH2CH(R7)COOH which can be conveniently reduced to an alcohol by reaction with isobutylchloroformate and reduction with sodium borohydride using suitable protection if necessary.
Such an alcohol can be reacted using suitable protection, to introduce the Lp group by reactions such as:
alkylation with an alkyl halide in the presence of a base;
reaction with diethyl azodicarboxylate/triphenylphosphine and a hydroxylated aryl compound;
by reaction with an activated carboxylic acid (e.g. an acid chloride) or with a carboxylic acid and diethylazodicarboxylate/triphenylphosphine;
by reaction with an isocyanate; and
by treatment with methanesulphonyl chloride or trifluoromethanesulphonic anhydride and reaction with an amine, or with a thiol optionally followed by oxidation, e.g. with potassium metaperiodate or hydrogen peroxide.
In this way compounds with linkages of xe2x80x94CH2xe2x80x94Oxe2x80x94, xe2x80x94CH2xe2x80x94Oxe2x80x94COxe2x80x94, xe2x80x94CH2xe2x80x94Oxe2x80x94COxe2x80x94NR1xe2x80x94, xe2x80x94CH2xe2x80x94NR1xe2x80x94, xe2x80x94CH2xe2x80x94Sxe2x80x94, xe2x80x94CH2xe2x80x94SOxe2x80x94 and xe2x80x94CH2xe2x80x94SO2xe2x80x94 between the alpha carbon and the lipophilic group may be produced. These compounds can then be coupled with 1-amino-7-isoquinoline carboxylic acid TFA salt.
Alternatively the alcohol can be oxidized to form a corresponding aldehyde (e.g. by oxidation with manganese dioxide or DMSO/oxalyl chloride or DMSO/SO3 or Dess-Martin reagent) which may be reacted to introduce the Lp group by reactions such as:
reaction with Wittig reagents or Horner-Emmons reagents, optionally followed by reduction of the resulting carbon:carbon double bond using H2/Pd-carbon;
reaction with an organometallic, eg a Grignard reagent, optionally followed by reaction on the resulting hydroxyl group, such as oxidation (eg with MnO2, DMSO/oxalyl chloride or Dess-Martin reagent), alkylation (eg with an alkyl halide in the presence of a base in a solvent such as DMF), arylation (eg with diethyl-azodicarboxylate/triphenyl phosphine and a hydroxyaryl compound), ester formation (eg with an acid chloride or with a carboxylic acid and diethylazido dicarboxylate/triphenyl phosphine), or carbamate formation (eg with an isocyanate);
by reaction with an amine followed by reduction, e.g. with sodium cyanoborohydride;
by reaction with a hydrazine; or
by reaction with a carbazide.
In this way compounds with linkages of xe2x80x94CHxe2x95x90CR1xe2x80x94, xe2x80x94CH2xe2x80x94CHR1xe2x80x94, xe2x80x94CHOHxe2x80x94, xe2x80x94CHR1xe2x80x94Oxe2x80x94, xe2x80x94CHR1xe2x80x94Oxe2x80x94COxe2x80x94, xe2x80x94CHR1xe2x80x94Oxe2x80x94COxe2x80x94NR1xe2x80x94, xe2x80x94COxe2x80x94, xe2x80x94CH2xe2x80x94NR1xe2x80x94, xe2x80x94CHxe2x95x90Nxe2x80x94NR1xe2x80x94 and xe2x80x94CHxe2x95x90Nxe2x80x94NR1xe2x80x94COxe2x80x94NR1xe2x80x94 between the alpha carbon and the lipophilic group may be produced. Again the resulting compounds could then be coupled to the isoquinoline derivative.
The transformation of alcohol to amine referred to above may be used to produce an amine reagent for lipophilic group introduction, e.g. a compound NH2xe2x80x94CH(R7)xe2x80x94CH2xe2x80x94NR1H.
Such an amine reagent may be reacted to introduce the lipophilic group, e.g. by acylation with an acid halide or activated ester, by reaction with isocyanate, by reaction with an isothiocyanate, or by reaction with a sulphonyl chloride. In this way compounds with linkages of xe2x80x94CH2NR1xe2x80x94COxe2x80x94, xe2x80x94CH2xe2x80x94NR1xe2x80x94COxe2x80x94NR1xe2x80x94, xe2x80x94CH2NR1xe2x80x94CSxe2x80x94NR1xe2x80x94 and -xe2x80x94H2NR1xe2x80x94SO2xe2x80x94 between the alpha carbon and the lipophilic groups may be produced.
The transformation of acid to amide referred to above may be used to produce an amide reagent for introduction of the Lp group, e.g. a compound NH2xe2x80x94CH(Ri)xe2x80x94CONH2.
Such amides may be reacted to introduce lipophilic groups, e.g. by reaction with a haloketone (e.g. phenacyl bromide). This provides a linkage 
from alpha carbon to lipophilic group.
Analogously the amide may be transformed to a thioamide by reaction with Lawesson""s reagent and then reacted with a haloketone to form a linkage 
The amide reagent may likewise be transformed to a nitrile reagent by dehydration, e.g. with trifluoroacetic anhydride. The nitrile reagent may be reacted with hydrazine then with acyl halide and then cyclized, (e.g. with trifluoroacetic anhydride) to produce a linkage 
Alternatively it may be treated with hydroxylamine then reacted with acyl halide and cyclized (e.g. with trifluoroacetic anhydride) to produce a linkage 
The hydrazide produced by reaction of a carboxylic acid reagent with hydrazine discussed above may likewise be used as a reagent for lipophilic group introduction, e.g. as a compound of formula NH2xe2x80x94CH(Ri)xe2x80x94COxe2x80x94NR1xe2x80x94N(R1)2.
Thus the hydrazide reagent can be reacted with an acyl halide and cyclized, e.g. with trifluoroacetic anhydride to yield a linkage 
or reacted with an acyl halide or an isocyanate to yield linkages xe2x80x94COxe2x80x94NRxe2x80x94NR1xe2x80x94COxe2x80x94 and xe2x80x94COxe2x80x94NR1xe2x80x94NR1xe2x80x94COxe2x80x94NR1xe2x80x94 respectively.
Alternatively the hydrazide may be transformed by reaction with Lawesson""s reagent and then reacted with an acyl halide and cyclized (e.g. with trifluoroacetic anhydride) to produce the linkage 
An alternative route to these compounds is to carry out any of the above chemical reactions to incorporate the lipophilic group (and optional H bond donor) into a protected intermediate such as a compound of formula (IV). 
The protecting group may then be removed before coupling of the 1-amino-7-carboxyisoquinoline (optionally protected).
A starting reagent for lipophilic group introduction where the alpha atom is nitrogen may be produced for example by reaction of a beta protected hydrazine (such protection to be chosen as to be compatible with the subsequent reagents to be employed) with phosgene, diphosgene, triphosgene or N,Nxe2x80x2carbonyl diimidazole to give a reactive compound of the type: 
This intermediate may be used as has been described above for the carboxylic starting reagents where the alpha atom is carbon.
Removal of the protecting group by standard methods and coupling with an activated 1-amino-7-carboxyisoquinoline will give compounds of the type
IQ-xe2x80x94CONHxe2x80x94N(R7)xe2x80x94Lxe2x80x94Lp(D)n
(where R7, L, Lp and D are as defined above and IQ is a 1-aminoisoquinoline linked in the 7-position).
Thus viewed from a further aspect the invention provides a process for the preparation of a compound according to the invention which process comprises coupling a carbonyl attached group to 1-amino-7-carboxyisoquinoline.
The compounds of the invention may be administered by any convenient route, e.g. into the gastrointestinal tract (e.g. rectally or orally), the nose, lungs, musculature or vasculature or transdermally. The compounds may be administered in any convenient administrative form, e.g. tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches etc. Such compositions may contain components conventional in pharmaceutical preparations, e.g. diluents, carriers, pH modifiers, sweeteners, bulking agents, and further active agents. Preferably the compositions will be sterile when in a solution or suspension form suitable for injection or infusion. Such compositions form a further aspect of the invention.
Viewed from this aspect the invention provides a pharmaceutical composition comprising a serine protease inhibitor according to the invention together with at least one pharmaceutically acceptable carrier or excipient.
Viewed from a further aspect the invention provides the use of a serine protease inhibitor according to the invention for the manufacture of a medicament for use in a method of treatment of the human or non-human animal body (e.g. a mammalian, avian or reptilian body) to combat a condition responsive to a serine protease inhibitor (e.g. a condition such as a thrombotic disorder responsive to a factor Xa inhibitor), said method comprising administering to said body an effective amount of a serine protease inhibitor according to the invention.
The dosage of the inhibitor compound of the invention will depend upon the nature and severity of the condition being treated, the administration route and the size and species of the patient. However in general, quantities of from 0.01 to 100 xcexcmol/kg bodyweight will be administered.
All publications referred to herein are hereby incorporated by reference.
The invention will now be described further with reference to the following non-limiting Examples.
Abbreviations used follow IUPAC-IUB nomenclature. Additional abbreviations are Hplc, high-performance liquid chromatography; DMF, dimethylformamide; DCM, dichloromethane; HAOt, 1-hydroxy-7-azabenzotriazole; HATU, [O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate]; Fmoc, 9-Fluorenylmethoxycarbonyl; HOBt, 1-hydroxybenzotriazole; TBTU, 2-(1H-(benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate; DIPEA, diisopropylethylamine; Boc, tertiary butyloxycarbonyl; DIPCI, diisopropylcarbodiimide; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; TEA, triethylamine; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; Rink linker, p-[(R,S)-xcex1-[1-(9H-Fluoren-9-yl)methoxyformamido]-2,4-dimethoxybenzyl]phenyl acetic acid; TFA, trifluoroacetic acid; MALDI-TOF, Matrix assisted laser desorption ionisationxe2x80x94time of flight mass spectrometry; and RT, retention time. Unless otherwise indicated amino acid derivatives, resins and coupling reagents were obtained from Novabiochem (Nottingham, UK) and other solvents and reagents from Rathburn (Walkerburn, UK) or Aldrich (Gillingham, UK) and were used without further purification. All solution concentrations are expressed as %Vol./%Vol. unless otherwise stated.
Purification
Purification was by gradient reverse phase Hplc on a Waters Deltaprep 4000 at a flow rate of 50 ml/min. using a Deltapak C18 radial compression is column (40 mmxc3x97210 mm, 10-15 mm particle size). Eluant A consisted of aqTFA (0.1%) and eluant B 90% MeCN in aqTFA(0.1%) with gradient elution (Gradient 1, 0 min. 20%B then 20% to 100% over 36 min., Gradient 2, 0 min. 5%B for 1 min. then 5%B to 20%B over 4 min., then 20% to 60% over 32 min. or Gradient 3, 0 min. 20%B then 20% to 100% over 15 min.). Fractions were analysed by analytical Hplc and MALDI-TOF or electrospray LCMS before pooling those with  greater than 95% purity for lyophilisation.
Analysis
Analytical Hplc was on a Shimadzu LC6 gradient system equipped with an autosampler, a variable wavelength detector at flow rates of 0.4 ml/ min. Eluents A and B as for preparative Hplc. Columns used were Techogell5 C18 (2xc3x97150 mm)(Hplc Technology), Magellan C8 column (2.1xc3x97150 mm, 5 mm particle size) (Phenomenex). Purified products were further analysed by MALDI-TOF and nmr.
Method 1
Using a solid phase strategy on a Protein Technologies, Symphony Multiple Peptide Synthesiser by attachment of bis amino compounds to Peg-2-chlorotrityl chloride resin: 2-Chlorotrityl chloride resin was typically treated with greater than 2 fold excess of the di-amine in dry DCM. The resin was further modified by the attachment of acids. Activation of Fmoc protected amino acid (2-5 eq) was by TBTU/ DIPEA, all couplings (minimum 120 min.) were carried out in DMF. Deprotection of the Fmoc group was achieved with 20% piperidine in DMF. Other acid substituents were added as the HOBt or HOAt esters either by activation with HBTU/HATU or DIPCI with or without Boc protection of amino groups. Cleavage of the products from the resin was by treatment (30 min., ambient) with 10% triethylsilane in TFA, filtration, evaporation and trituration with diethylether.
Synthesis Using the Symphony Multiple Peptide Synthesiser
The Symphony Multiple Peptide Synthesiser is charged with DMF, DCM, TBTU in DMF(450 mM), DIPEA in DMF (900 mM), 20% piperidine in DMF. Resins are held in plastic reaction vessels that allow the introduction of reagents and solvents and nitrogen for agitation or air drying.
A typical synthesis cycle on the Symphony is as follows:
The reaction vessel containing the resin (0.1 mmol) is charged with the Fmoc protected amino acid (0.5 mmol) and then this is dissolved in DMF (2.5 ml), treated with TBTU (056 mmol, 1.25 ml) and DIPEA (1.1 mmol, 1.25 ml) and agitated with nitrogen for 2 hours (agitation times may vary). After coupling, the resin is washed with DMF (6xc3x975 ml) then deprotected with 20% piperidine in DMF (2xc3x975 ml for 1 min. each, then 1xc3x975 ml for 8 min.). The resin is then washed with DMF (6xc3x975 ml).